DESCRIPTION (provided by applicant Protein modification with the small protein ubiquitin, a process referred to as ubiquitylation, plays crucial roles in the majority of cellular processes. Ubiquitin ligases are the most complex and most important components of the ubiquitylation machinery. They confer substrate selectivity and are the main targets for regulation. The importance of ubiquitin ligases (E3 enzymes) is underscored by the vast number of proteins functioning as E3s. It is estimated that the human genome encodes 600- 1000 proteins with ubiquitin ligase activity, a number that is significantly higher than that of protein kinases. Ubiquitin ligases are poised to form a link between metabolic pathways and other cellular processes such as cell cycle, stress response, and differentiation. The importance of cross talk between metabolism and other cellular pathways is evident, and it is becoming increasingly clear that a plethora of human diseases is directly connected to misregulation at the interface between metabolism and signaling to other cellular pathways. Despite the importance of understanding how metabolism communicates with other cellular processes, our understanding at the molecular level is very limited at best. The parent grant of this proposal uses a pathway that connects sulfur amino acid metabolism with cell proliferation as a model to understand regulation by non-proteolytic ubiquitylation. The central player is the ubiquitin ligase SCFMet30, which integrates metabolism of sulfur containing metabolites with the cell cycle. This revision application proposes to significantly extend the scope of the parent grant to understand how levels of sulfur containing metabolites regulate the SCFMet30 ubiquitin ligase (specific aim 1), and to explore the hypothesis that ubiquitin ligases directly connect metabolic pathways with other cellular processes (specific aim 2). We will develop and apply mass spectrometric approaches to probe interactions of metabolites with components of the SCFMet30 pathway (specific aim 1), and other ubiquitin ligases in yeast and human cells (specific aim 2). The physiological importance of identified interactions will then be probed using mutations in the identified binding sites. Ubiquitin ligases are the most diverse group of cellular regulators and exciting findings in the plant system have demonstrated ubiquitin ligases as receptors for metabolite related small molecules. This proposal aims to define ubiquitin ligases as the molecular link between metabolism and other cellular processes in yeast and humans. Findings from these studies are likely to define new paradigms in metabolite sensing and to uncover novel disease related pathways.